Apoptogenic activity of 2 alpha,3 alpha-dihydroxyurs-12-ene-28-oic acid from Prunella vulgaris var. lilacina is mediated via mitochondria-dependent activation of caspase cascade regulated by Bcl-2 in human acute leukemia Jurkat T cells

作者：Woo Hyun Ju; Jun Do Youn; Lee Ji Young; Woo Mi Hee; Yang Chae Ha; Kim Young Ho^*

来源：Journal of Ethnopharmacology, 2011, 135(3): 626-635.

DOI：10.1016/j.jep.2011.03.067

• Prunella vulgaris var. lilacina
• 2 alpha,3 alpha-Dihydroxyurs-12-en-28-oic acid
• Mitochondrial damage
• Caspase cascade
• Apoptosis
• Human Jurkat T cells

Ethnopharmacological relevance: The dried spikes of Prunella vulgaris var. lilacina (Labiatae) have been used for traditional herbal medicine to treat fever, inflammation, dropsy, gonorrhea and cancer in Korea, Japan and China. The present study evaluated the apoptotic effect of 2 alpha,3 alpha-dihydroxyurs-12-en-28-oic acid (DHURS), purified from the dried spikes on human acute leukemia Jurkat T cells.
Materials and methods: Cell viability was assessed by MTT assay. Mitochondrial membrane potential (Delta psi m) loss, apoptotic change of the cell cycle, and apoptotic cells were measured by flow cytometric analysis. Mitochondrial cytochrome c release and activation of caspase cascade were determined by Western blot analysis. Caspase-12 activity and caspase-3 activity were assayed using the Fluorometric Assay Kit and the Colorimetric Assay Kit, respectively.
Results: Treatment of Jurkat T cells with DHURS (20-25 mu g/ml) caused cytotoxicity and apoptotic DNA fragmentation along with Delta psi m loss, mitochondrial cytochrome c release, activation of caspase-9, -7, -3, and -8, and PARP degradation. However, these apoptotic events were abrogated by overexpression of Bcl-2. Pretreatment of the cells with the pan-caspase inhibitor (z-VAD-fmk), the caspase-9 inhibitor (z-LEHD-fmk) or the caspase-3 inhibitor (z-DEVD-fmk) to prevent DHURS-induced apoptosis could block the activation of caspase-7 and -8, and PARP degradation, but not the Delta psi m loss, activation of caspase-9 and -3. Both FADD- and caspase-8-positive wild-type Jurkat clone A3, FADD-deficient Jurkat clone 12.1, and caspase-8-deficient Jurkat clone 19.2 exhibited similar susceptibilities to the cytotoxicity of DHURS, excluding an involvement of Fas/FasL system in triggering the apoptosis. The IC(50) value for Jurkat T cells was similar to 22 mu g/ml, whereas that for human peripheral T cells was 25 mu g/ml.
Conclusions: These results indicate that DHURS-induced apoptogenic activity in Jurkat T cells, which was less potent in normal peripheral T cells, was mediated by Delta psi m loss, mitochondrial cytochrome c release, and subsequent activation of caspase-9 and -3, leading to activation of caspase-7 and -8, which could be regulated by Bcl-2.

• 出版日期2011-6-1